PROJECT SUMMARY Deregulated intracellular protein interactions mediate a complex network of pathologic signaling events that drive human cancer. The development of drugs to modulate the large, flat, and complex interfaces of oncogenic signaling proteins remains a formidable challenge and has inspired alternative approaches to traditional small molecule discovery. Although the very peptide structures that define the molecular handshakes between proteins are ideally suited to modulate such signaling events, structured peptides in the context of a protein typically lose their bioactive shape when isolated from the whole. We previously developed hydrocarbon-stapled peptides that recapitulate the natural shape of ?-helical interaction motifs by insertion of chemical crosslinks. Stapled peptides are structurally-stable, protease-resistant, and retain the capacity to engage their biological targets with natural potency and specificity. Unexpectedly, select stapled ?-helical peptides are also cell- permeable, opening the door to an entirely new modality for dissecting and potentially drugging pathologic protein interactions in cancer. However, despite a decade of progress in the creation of new research tools to investigate and target disease-causing proteins, the true promise of structured peptides as a novel therapeutic platform for cancer has been hampered by our very limited understanding of two fundamental questions: (1) What biophysical properties dictate whether a stapled peptide will be taken up by a cancer cell? (2) What is the explicit molecular mechanism of cellular import and intracellular release for cell-permeable stapled peptides? The answers to these questions not only carry the potential to transform these research tools and prototype therapeutics into an arsenal of bona fide cancer drugs, but will also provide critical new insight into the essential cellular process of vesicle transport. For example, certain cancer cells enforce metabolic immortality by upregulating protein nutrient uptake via the pinocytosis import pathway ? a mechanism that could be leveraged to achieve a therapeutic window for structured peptide-based cancer drugs. Here, I propose a two-pronged research plan that harnesses our deep experience with stapled peptide design and application, but diverges from our traditional protein targeting research to instead focus on the why and how distinct stapled peptides access the intracellular environment. To achieve our goals, we will (1) employ biostatistical and computational methods to glean what biophysical parameters confer cellular penetrance among our libraries of stapled peptides, and (2) perform a genome-wide CRISPR-based screen to identify and then vet those cellular components that alternatively impair and enhance the import pathway for stapled peptides. By integrating our laboratory?s foundation in the chemical biology of oncogenic protein interactions with the proposed biostatistical, CRISPR screening, and cancer cell import validation studies, we hope to break new ground in our understanding of just how stapled peptides and their uptake mechanisms can be harnessed for therapeutic benefit in cancer.